Guanosine 5'-triphosphate cyclohydrolase I (GTPCH1) is a homodecameric protein consisting of 25-kDa subunits, which enzyme catalyzes the rearrangement of GTP to dihydroneopterin triphosphate, a species subsequently converted to tetrahydrobiopterins (BH4) through the sequential action of 6-pyruvoyltetrahydrobiopterin synthase and sepiapterin reductase. In contrast to the latter two enzymes, GTPCH1 is the rate-limiting enzyme in most tissues, making it the major determinant of intracellular BH4 content. GTPCH1 is constitutively expressed in vascular cells; however, whether GTPCH1 is critical for the maintenance of BH4 levels in these cells is unknown. Several recent studies suggest that BH4 deficiency is responsible for endothelial nitric oxide synthase (eNOS) uncoupling during hypertension, as seen by the finding that hypertension and related eNOS uncoupling are effectively prevented by co- administration of sepiapterin, a precursor for BH4. BH4 supplementation or increased BH4 synthesis by GTPCH1 restores BH4 levels and normalizes eNOS function in the deoxycorticosterone acetate (DOCA)-salt hypertensive mice. GTPCH1 activity, as well as BH4 levels, is reduced in these mice. However, the in vivo cause-effect relationship between eNOS uncoupling and GTPCH1, which is critical for eNOS-mediated protection of endothelial function, has yet to be investigated. In particular, it is unclear how pathological stimuli such as hypertension reduce GTPCH1 levels and how stimuli such as angiotensin-II (Ang II) and hypertension modulate proteasome function. Our exciting new preliminary data have led us to hypothesize that Ang II, via oxidants such as ONOO-, increases 26S proteasome-mediated degradation of GTPCH1, resulting in BH4 deficiency, eNOS uncoupling, and the elevation of blood pressure. This central hypothesis will be tested in three interrelated specific aims by using a combination of experimental approaches including purified proteins, cultured cells, and several models of hypertension in vivo. The proposed studies are significant, as they will deepen our understanding of the upstream regulation of BH4 levels, the contribution of oxidative stress and ubiquitin-proteasome systems in the development of vascular injury, and the contribution of oxidative stress in blood pressure regulation. In particular, these studies will provide clues as to the therapeutic effects of MG132, a proteasome inhibitor that was recently approved by the FDA for cancer therapy.